Device, system, and method including micro-patterned cell treatment array

ABSTRACT

Devices, systems, or methods are disclosed herein for treatment of disease in a vertebrate subject. The device can include a quasi-planar substrate; and one or more laterally-mobile effector molecule types at least partially embedded within the quasi-planar substrate, wherein the one or more laterally-mobile effector molecule types is configured to interact with one or more cell types. The device can further include one or more sensors configured to detect at least one aspect of an interaction between the at least one of the one or more laterally-mobile effector molecule types and the one or more cell types; and a controller in communication with the one or more sensors, wherein the controller is configured to responsively initiate modification of at least one of the one or more laterally-mobile effector molecule types, the quasi-planar substrate, and the one or more cell types.

SUMMARY

Devices, systems, and methods are disclosed herein for treatment ofdisease involving one or more cell types in a vertebrate subject. Thedevice can responsively interact with the one or more cell types tomodify or differentiate the one or more cell types in the vertebratesubject, or to induce apoptosis or necrosis in the one or more celltypes in the vertebrate subject. The cell types can include one or moreof immune cells, pathogens, pathologic cells, tumor cells, neoplasticcells, or defective cells. A device is disclosed that includes aquasi-planar substrate; and one or more laterally-mobile effectormolecule types at least partially embedded within the quasi-planarsubstrate, wherein at least one of the one or more laterally-mobileeffector molecule types is configured to interact with one or more celltypes. The device can further include one or more sensors configured todetect at least one aspect of an interaction between the at least one ofthe one or more laterally-mobile effector molecule types and the one ormore cell types; and a controller in communication with the one or moresensors, wherein the controller is configured to responsively initiatemodification of at least one of the one or more laterally-mobileeffector molecule types, the quasi-planar substrate, and the one or morecell types. The device can responsively interact with eukaryotic cellsor prokaryotic cells to modify, differentiate, or kill the cells.

At least one of the one or more effector molecule type can include, butis not limited to, a ligand molecule, a receptor molecule, or acytokine. The one or more laterally-mobile effector molecule typesmodified in response to the controller can be configured to be differentfrom the one or more laterally-mobile effector molecule types sensedinteracting with the one or more cell types by the one or more sensors.The at least two of the one or more laterally-mobile effector moleculetypes can be configured such that the one or more cell types interactsequentially with the at least two of the one or more laterally-mobileeffector molecule types. The at least two of the one or morelaterally-mobile effector molecule types can be configured such that theone or more cell types interact simultaneously with the at least two ofthe one or more laterally-mobile effector molecule types. The controllercan be configured to responsively add at least one of the one or morelaterally-mobile effector molecule types to the quasi-planar substrate.The controller can be configured to responsively remove at least one ofthe one or more laterally-mobile effector molecule types from thequasi-planar substrate. The quasi-planar substrate can include one ormore discreet regions configured to include at least one of the one ormore laterally-mobile effector molecule types. The controller can beconfigured to responsively move at least one of the one or morelaterally-mobile effector molecule types and the one or more cell typesrelative to the quasi-planar substrate. The controller can be configuredto responsively remove at least one of the one or more laterally-mobileeffector molecule types and the one or more cell types from thequasi-planar substrate.

The quasi-planar substrate can include a liquid configured as aninterface with a cell type-carrying fluid. The liquid can be configuredto at least partially embed the one or more laterally-mobile effectormolecule types on the quasi-planar substrate. The quasi-planar substratecan include a membrane. The membrane can be bordered by a solidsubstrate. The membrane can include a lipid bilayer. The membrane caninclude a lipid monolayer. The membrane can be configured to contact aliquid or solid surface within the quasi-planar substrate. The celltype-carrying fluid can include, but is not limited to, a gas or aliquid. The quasi-planar substrate can include a solid surfaceconfigured to at least partially embed at least one of the one or morelaterally-mobile effector molecule types. The quasi-planar substrate caninclude a liquid at least partially surrounding the solid surface,wherein the liquid is configured to at least partially embed at leastone of the one or more laterally-mobile effector molecule types withinthe liquid. The quasi-planar substrate can be configured to mobilize atleast one of the one or more laterally-mobile effector molecule types inessentially two dimensions on the quasi-planar substrate. Thequasi-planar substrate can be configured to bi-directionally mobilize atleast one of the one or more laterally-mobile effector molecule types inessentially one dimension on the quasi-planar substrate. Thequasi-planar substrate can be configured to include one or morespatially-discrete portions. At least one of the one or morespatially-discrete portions can include two or more identical of the oneor more laterally-mobile effector molecule type. At least one of the oneor more spatially-discrete portions can include two or more different ofthe one or more laterally-mobile effector molecule type. The one or morespatially-discrete portions can include one or more of array sites orchannels. The one or more spatially-discrete portions can be connectedwith a fluidic interface. The controller can include one or more ofstatic connections or dynamic connections, wherein the one or more ofthe static connections or the dynamic connections are configured tocontrol communication between the one or more spatially discreteportions. The one or more of the static connections or the dynamicconnections can be fluid connections.

The one or more laterally-mobile effector molecule can include, but isnot limited to, one or more of protein, oligonucleotide, or aptamer. Theone or more laterally-mobile effector molecule can include, but is notlimited to, one or more of antibody, B cell receptor, T cell receptor,major histocompatibility complex (MHC) protein, antigen-loaded WICprotein, co-stimulatory molecule, or cytokine. The interaction betweenthe one or more laterally-mobile effector molecule types and the one ormore cell types can include interaction with a receptor on the one ormore cell types. At least one of the one or more laterally-mobileeffector molecule types can be configured to undergo a conformationalchange in response to interaction with the one or more cell types. Atleast one of the one or more laterally-mobile effector molecule typescan be configured to react with one or more binding components inresponse to interaction with the one or more cell types. The one or morebinding components can include, but is not limited to, protein,antibody, receptor, ligand, major histocompatibility complex (WIC),antigen-loaded WIC, co-stimulatory molecule, oligonucleotide, aptamer,or cytokine. The one or more binding components can be laterally mobilein the substrate. The one or more binding components can be mobile in aliquid of the substrate. The one or more binding components can be acomponent of intracellular signaling. The one or more binding componentscan be on the one or more cell types. At least one of the one or morelaterally-mobile effector molecule types can be configured to bind andinteract with a single cell type of the one or more cell types. Thecontroller can be configured to responsively interact with the singlecell type. The single cell type can include, but is not limited to, oneor more of T cells, B cells, dendritic cells, NK cells, macrophages,phagocytes, monocytes, lymphocytes, macrophages, neutrophils,granulocytes, eosinophils, basophils, mast cells, stem cells, orgerm-line cells. The controller can be configured to responsively modifythe single cell type. The controller can be configured to responsivelymodify the single cell type by one or more of activating B cells,activating T cells, changing cell status to memory cell or from memorycell, altering cytokine expression profiles, inducing affinitymaturation, inducing hyperaffinity maturation, or inducing or changingstem cell differentiation. The controller can be configured toresponsively modify the single cell type by inducing anergy, removinganergy, inducing apoptosis, or inducing necrosis. The sensor can beconfigured to identify the single cell type, and the controller can beconfigured to modify the single cell type. The one or morelaterally-mobile effector molecules can include a fluorescent component.

The one or more sensors can include, but is not limited to, a masssensor, force sensor, weight sensor, or surface vibration sensor. Theone or more sensors can include, but is not limited to, optical sensor,plasmonic sensor, electrical sensor, capacitive sensor, or chemicalsensor. The one or more sensors can be configured to spatially detect atleast one of the one or more laterally-mobile effector molecule typesand at least one of the one or more cell types in one or morespatially-discrete portions of the quasi-planar substrate. The one ormore sensors can be configured to detect one or more interactions of atleast one of the one or more laterally-mobile effector molecule typeswith at least one of the one or more cell types, wherein the one or moreinteractions occur at the one or more spatially-discrete portions of thequasi-planar substrate. The one or more sensors can be configured todetect a position of at least one of the one or more cell types bound tothe quasi-planar substrate. The one or more sensors can be configured todetect a position of the one or more laterally-mobile effector moleculetypes on the quasi-planar substrate. The controller can be configured torespond in one or more of a real-time response, in a delayed response,in a sequenced response, or in a response dependent upon a combinationof previously-sensed interactions. At least one of the one or morelaterally-mobile effector molecule types can be configured to increasebinding of at least one of the one or more cell types to thequasi-planar substrate. At least one of the one or more laterally-mobileeffector molecule types can be configured to decrease binding of atleast one of the one or more cell types to the quasi-planar substrate.

The controller can be configured to direct motion of at least one of theone or more laterally-mobile effector molecule types toward or away fromat least one of the one or more cell types. The controller can beconfigured to interact with at least one of a magnetic component, anelectrical component, or an optical component of the one or morelaterally-mobile. The controller can be configured to respond byintroducing one or more components into or onto at least one of the oneor more cell types. The controller can be configured to respond byintroducing one or more components into or onto at least one of the oneor more cell type by endocytosis, transfection, transformation,electroporation, diffusion, pore transport, active membrane transport,passive membrane transport, or membrane-to-membrane transfer. The one ormore components can be configured to allow at least one of the one ormore cell type to be subsequently acted upon, tracked, identified, orkilled. The one or more components can include one or more of geneticmaterial, chemical entity, nucleic acid, protein, lipid, virus, vesicle,detectable tag, QDOT, or magnetic nanoparticle. The controller can beconfigured to respond by applying energy in the form of one or more oflight, heat, magnetism, pressure, electricity, or vibration. Thecontroller can be configured to respond by applying fluidic force ormotional force to the one or more laterally-mobile effector moleculetypes or the quasi-planar substrate. The force can be transferred to thecell by one or more bound laterally-mobile effector molecule types. Thecontroller can be configured to respond by applying the fluidic force orthe motional force utilizing one or more of outflow pump, intake pump,surface tension, electrical force, laser light-induced force, ormagnetic force. The controller can be configured to respond by applyingmotional force to the one or more cell types. The controller can beconfigured to respond by altering connectivity of the quasi-planarsubstrate. The controller can be configured to respond by altering MEMSbarriers on the quasi-planar substrate. The controller can be configuredto move at least one of the one or more cell types between one or morespatially discrete portions of the quasi-planar substrate. The one ormore spatially discrete portions of the quasi-planar substrate can beconfigured to perform one or more actions on at least one of the one ormore cell types, expose the one or more cell types to differentenvironments, or collect the one or more cell types into a common site.The controller can be configured to rotate at least one of the one ormore cell types to a new orientation. The controller can be configuredto rotate at least one of the one or more cell types by differentialbinding or release at one or more different sites on the at least one ofthe one or more cell types. The controller can be configured to exposedifferent cell regions or cell receptors to the quasi-planar substrate.The controller can be configured to respond to binding of at least oneof the one or more cell types at one region of the quasi-planarsubstrate by altering a configuration of the one or morelaterally-mobile effector molecule types at a second region on thequasi-planar substrate. The device can further include one or more fluidbypasses configured to receive and process at least one of the one ormore cell types in one or more fluids from a vertebrate subject throughthe device and configured to return processed fluids through the one ormore fluid bypasses into the vertebrate subject.

A method is disclosed that includes embedding one or morelaterally-mobile effector molecule types at least partially within aquasi-planar substrate of a device, transferring one or more cell typesto the quasi-planar substrate of the device, interacting at least one ofthe one or more laterally-mobile effector molecule types with the one ormore cell types, sensing at least one aspect of an interaction betweenthe one or more laterally-mobile effector molecule types and the one ormore cell types, and controlling, in response to the sensing, initiationof modification of at least one of the one or more laterally-mobileeffector molecule types, the quasi-planar substrate, and the one or morecell types. At least one of the one or more effector molecule type caninclude, but is not limited to, a ligand molecule, a receptor molecule,or a cytokine. The one or more laterally-mobile effector molecule typesmodified in response to the controller can be configured to be differentfrom the one or more laterally-mobile effector molecule types sensedinteracting with the one or more cell types by the one or more sensors.

The method can include interacting the one or more cell types with atleast two of the one or more laterally-mobile effector molecule typessuch that the one or more cell types interact sequentially with the atleast two of the one or more laterally-mobile effector molecule types.The method can include interacting the one or more cell types with atleast two of the one or more laterally-mobile effector molecule typessuch that the one or more cell types interact simultaneously with the atleast two of the one or more laterally-mobile effector molecule types.In the method, controlling movement can include adding at least one ofthe one or more laterally-mobile effector molecule types to thequasi-planar substrate. The quasi-planar substrate can include one ormore discreet regions configured to include at least one of the one ormore laterally-mobile effector molecule types. In the method,controlling movement can include removing at least one of the one ormore laterally-mobile effector molecule types from the quasi-planarsubstrate. In the method, controlling movement can include moving atleast one of the one or more laterally-mobile effector molecule typesand the one or more cell types on the quasi-planar substrate. In themethod, controlling movement can include removing at least one of theone or more laterally-mobile effector molecule types and the one or morecell types from the quasi-planar substrate.

In the method, the quasi-planar substrate can include a liquidconfigured as an interface with a cell type-carrying fluid. The methodcan include embedding at least one of the one or more laterally-mobileeffector molecule types in the liquid of the quasi-planar substrate. Thequasi-planar substrate can include a membrane. The membrane can bebordered by a solid substrate. The method can include contacting aliquid surface or a solid surface of the quasi-planar substrate with themembrane. The method can include mobilizing at least one of the one ormore laterally-mobile effector molecule types in essentially twodimensions on the quasi-planar substrate. The method can includebi-directionally mobilizing at least one of the one or morelaterally-mobile effector molecule types in essentially one dimension onthe quasi-planar substrate. The quasi-planar substrate can be configuredto include one or more spatially-discrete portions. In the method, eachof the one or more spatially-discrete portions can include two or moreidentical of the one or more laterally-mobile effector molecule type. Inthe method, each of the one or more spatially-discrete portions caninclude two or more different of the one or more laterally-mobileeffector molecule type. The one or more spatially-discrete portions caninclude one or more of array sites or channels. The method can includefluidically connecting the one or more spatially-discrete portions. Themethod can include controlling communication between the one or morespatially discrete portions with one or more of static connections ordynamic connections. The one or more of static connections or thedynamic connections can be fluid connections.

The method can include inducing a conformational change in at least oneof the one or more laterally-mobile effector molecule types in responseto interaction with the one or more cell types. In the method, at leastone of the one or more laterally-mobile effector molecule types can beconfigured to bind and interact with a single cell type of the one ormore cell types. The method can include identifying the single cell typeby one or more sensors, and modifying the single cell type in responseto action of the controller. The method can include spatially detectingat least one of the one or more laterally-mobile effector molecule typesand the one or more cell types in one or more spatially-discreteportions of the quasi-planar substrate with one or more sensors. Themethod can include detecting one or more responsive interactions of atleast one of the one or more laterally-mobile effector molecule typeswith the one or more cell types by the one or more sensors, wherein theone or more responsive interactions occur at the one or morespatially-discrete portions of the quasi-planar substrate. The methodcan include detecting a position of the one or more cell types bound tothe quasi-planar substrate by the one or more sensors. The method caninclude introducing at least one of the one or more laterally-mobileeffector molecule types to the quasi-planar substrate by response of thecontroller. The method can include removing at least one of the one ormore laterally-mobile effector molecule types from the quasi-planarsubstrate by response of the controller. In the method, at least one ofthe one or more laterally-mobile effector molecule types can beconfigured to increase binding of the one or more cell types to thequasi-planar substrate. In the method, at least one of the one or morelaterally-mobile effector molecule types can be configured to decreasebinding of the one or more cell types to the quasi-planar substrate. Inthe method, the controlling can be configured to direct motion of atleast one of the one or more laterally-mobile effector molecule typestoward or away from the one or more cell types. In the method, thecontrolling can be configured to direct application of fluidic force ormotional force to at least one of the one or more laterally-mobileeffector molecule types or the quasi-planar substrate.

In the method, the controlling can be configured to direct alteration ofconnectivity of the quasi-planar substrate. In the method, thecontrolling can be configured to direct alteration of MEMS barriers onthe quasi-planar substrate. In the method, the controlling can beconfigured to direct alteration of movement the one or more cell typesbetween one or more spatially discrete portions of the quasi-planarsubstrate. In the method, the controlling can be configured to directalteration of rotation of the one or more cell types to a neworientation. In the method, the controlling can be configured to respondto binding of the one or more cell types at one region of thequasi-planar substrate by directing alteration of a configuration of atleast one of the one or more laterally-mobile effector molecule types ata second region on the quasi-planar substrate. The method can includereceiving and processing one or more cell types in one or more fluidsfrom a vertebrate subject through one or more fluid bypasses of thedevice and returning processed fluids through the one or more fluidbypasses into the vertebrate subject through the one or more fluidbypasses.

A system is disclosed that includes a fluid bypass configured to passagefluids including one or more cell types from a vertebrate subject into adevice and configured to return the passaged fluids to the vertebratesubject, wherein the device includes a quasi-planar substrate; and oneor more laterally-mobile effector molecule types at least partiallyembedded within the quasi-planar substrate, wherein the one or morelaterally-mobile effector molecule types is configured to interact withone or more cell types; one or more sensors configured to detect the atleast one aspect of the interaction between the one or morelaterally-mobile effector molecule types and the one or more cell types;and a controller in communication with the one or more sensors, whereinthe controller is configured to responsively initiate modification of atleast one of the one or more laterally-mobile effector molecule types,the quasi-planar substrate, and the one or more cell types. At least oneof the one or more effector molecule type can include, but is notlimited to, a ligand molecule, a receptor molecule, or a cytokine. Theone or more laterally-mobile effector molecule types modified inresponse to the controller can be configured to be different from theone or more laterally-mobile effector molecule types sensed interactingwith the one or more cell types by the one or more sensors.

The at least two of the one or more laterally-mobile effector moleculetypes can be configured such that the one or more cell types interactsequentially with the at least two of the one or more laterally-mobileeffector molecule types. The at least two of the one or morelaterally-mobile effector molecule types can be configured such that theone or more cell types interact simultaneously with the at least two ofthe one or more laterally-mobile effector molecule types. The controllercan be configured to responsively add at least one of the one or morelaterally-mobile effector molecule types to the quasi-planar substrate.The controller can be configured to responsively remove at least one ofthe one or more laterally-mobile effector molecule types from thequasi-planar substrate. The quasi-planar substrate can include one ormore discreet regions configured to include at least one of the one ormore laterally-mobile effector molecule types. The controller can beconfigured to responsively move at least one of the one or morelaterally-mobile effector molecule types and the one or more cell typesrelative to the quasi-planar substrate. The controller can be configuredto responsively remove at least one of the one or more laterally-mobileeffector molecule types and the one or more cell types from thequasi-planar substrate.

The quasi-planar substrate can include a liquid configured as aninterface with a cell type-carrying fluid. The liquid can be configuredto at least partially embed the one or more laterally-mobile effectormolecule types on the quasi-planar substrate. The quasi-planar substratecan include a membrane. The membrane can be bordered by a solidsubstrate. The membrane can include a lipid bilayer. The membrane caninclude a lipid monolayer. The membrane can be configured to contact aliquid or solid surface within the quasi-planar substrate.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C depict a diagrammatic view of an aspect of anembodiment of a device.

FIG. 2 depicts a logic flowchart of a method for treating a disease orcondition in a vertebrate subject.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

Devices, systems, and methods are disclosed herein for treatment ofdisease involving one or more cell types in a vertebrate subject. Thedevice can responsively interact with the one or more cell types tomodify or differentiate the one or more cell types in the vertebratesubject, or to induce apoptosis or necrosis in the one or more celltypes in the vertebrate subject. The cell types can include one or moreof immune cells, pathogens, pathologic cells, tumor cells, neoplasticcells, or defective cells. A device is disclosed that includes aquasi-planar substrate; and one or more laterally-mobile effectormolecule types at least partially embedded within the quasi-planarsubstrate, wherein at least one of the one or more laterally-mobileeffector molecule types is configured to interact with one or more celltypes. The device can further include one or more sensors configured todetect at least one aspect of an interaction between the at least one ofthe one or more laterally-mobile effector molecule types and the one ormore cell types; and a controller in communication with the one or moresensors, wherein the controller is configured to responsively initiatemodification of at least one of the one or more laterally-mobileeffector molecule types, the quasi-planar substrate, and the one or morecell types. The device can responsively interact with eukaryotic cellsor prokaryotic cells to modify, differentiate, or kill the cells.

The device includes a quasi-planar substrate that can incorporate one ormore fixed or laterally-mobile effector molecules that bind to cellsurface components on the one or more cell types. The quasi-planarsubstrate can include a liquid configured as an interface with a celltype-carrying fluid. The liquid can be configured to at least partiallyembed the one or more laterally-mobile ligand types. The liquid caninclude a membrane, e.g., a lipid bilayer or a lipid monolayer. Themembrane can be configured to contact a liquid or solid surface of thequasi-planar substrate. The laterally-mobile effector molecule types caninclude one or more of ligand molecules, receptor molecules, orcytokines. The device includes one or more sensors configured to detectthe one or more laterally-mobile effector molecule types and the one ormore cell types and can detect cell binding to at least one of the oneor more laterally-mobile effector molecule types on the substrate. Theone or more sensors can signal to a controller configured toresponsively initiate movement of the one or more laterally-mobileeffector molecule types and the one or more cell types in thequasi-planar substrate. The controller can responsively interact withthe sensor to modify the one or more cell types by sequentially exposingat least one of the laterally-mobile effector molecules to the one ormore cell types. The device includes a quasi-planar solid support andmultiple discreet regions or multiple reaction compartments on thequasi-planar solid support. The device includes a fluid media with atleast partially embedded laterally-mobile effector molecules, e.g., oneor more of ligand molecules, receptor molecules, or cytokines, andsensors to detect interaction of the at least partially embeddedlaterally-mobile effector molecules with the one or more cell types. Anexternal media in contact with the laterally-mobile effector moleculesand the sensors on the quasi-planar solid support is configured totransport the one or more cell types between reaction compartments. Theinterface between the quasi-planar solid support and the fluid media canbe maintained, e.g., by gravity, by surface tension, or by liquid-liquidimmiscibility.

The device disclosed herein can be used to modify functional cells,e.g., immune cells, effector cells, or receptor cells, in a vertebratesubject or to inactivate or kill pathogens, pathologic cells, tumorcells, neoplastic cells, or defective cells in a vertebrate subject.Cell refers to any cell or cells, as well as viruses, virus-infectedcells, or other particles having a size that is similar to that of abiological cell, including any prokaryotic cell, e.g., bacteria, oreukaryotic cell, e.g., fungi, plant cells or animal cells. Cells cantypically be spherical, or can be elongated, flattened, deformable orasymmetrical, i.e., not spherical. The size or diameter of a celltypically ranges from about 0.1 μm to 120 μm, or is from about 1 μm to50 μm. Cells modified by the device as described herein can be usefulfor immunotherapy, cancer therapy, anti-infective agents, regenerativemedicine, transplantation, reproductive biology, drug discovery, genediscovery, and other clinical, biotechnology, or research uses. Forimmunotherapy, the device as described herein can be used todifferentiate, mature, activate, induce anergy, load antigen,electroporate or transduce cells. Immune cells can be T lymphocytes, Blymphocytes, natural killer (NK) cells, lymphokine-activated killer(LAK) cells, dendritic cells, macrophages, monocytes, granulocytes,Kupffer cells, Langerhans cells, and other immune cell types. Tlymphocytes activated by the device can be helper T cells (T_(H),T_(H)1, T_(H)2, T_(H)17), cytolytic T cells, or regulatory T cells. Forexample, T_(H) cells can be activated by laterally mobile membraneproteins protruding from a lipid bilayer of the device. A method isdisclosed that includes embedding one or more laterally-mobile effectormolecule types at least partially within a quasi-planar substrate of adevice, transferring one or more cell types to the quasi-planarsubstrate of the device responsively interacting at least one of the oneor more laterally-mobile effector molecule types sequentially with theone or more cell types, wherein at least one of the one or morelaterally-mobile effector molecule types is configured to interactsequentially with the one or more cell types sensing at least one of theone or more laterally-mobile effector molecule types interacting withthe one or more cell types, and responsively controlling movement of atleast one of the one or more laterally-mobile effector molecule typesand the one or more cell types in the quasi-planar substrate.

A system is disclosed that includes a fluid bypass configured to passagefluids including one or more cell types from a vertebrate subject into adevice and configured to return the passaged fluids to the vertebratesubject, wherein the device includes a quasi-planar substrate; and oneor more laterally-mobile effector molecule types at least partiallyembedded within the quasi-planar substrate, wherein at least one of theone or more laterally-mobile effector molecule types is configured tointeract with one or more cell types; one or more sensors configured todetect at least one aspect of an interaction between the at least one ofthe one or more laterally-mobile effector molecule types and the one ormore cell types; and a controller in communication with the one or moresensors, wherein the controller is configured to responsively initiatemodification of at least one of the one or more laterally-mobileeffector molecule types, the quasi-planar substrate, and the one or morecell types.

With reference to the figures, and with reference now to FIGS. 1 and 2depicted is one aspect of a device, a method, or a system that may serveas an illustrative environment of and/or for subject mattertechnologies, for example, a device including a quasi-planar substrate;and one or more laterally-mobile effector molecule types embedded withinthe quasi-planar substrate, wherein the one or more laterally-mobileeffector molecule types is configured to responsively interact with oneor more cell types, and the one or more cell types interactssequentially with one or more of the one or more laterally-mobileeffector molecule types. Accordingly, the present disclosure describescertain specific devices or systems of FIGS. 1 and 2; and describesembodiments including certain specific devices, methods, and systems.Those having skill in the art will appreciate that the specific devicesor systems described herein are intended as merely illustrative of theirmore general counterparts.

Referring to FIG. 1A, 1B, and 1C, depicted is a partial diagrammaticview of one aspect of an embodiment of a device 100 including aquasi-planar substrate 110; and one or more laterally-mobile effectormolecule types 120, 130, 140 embedded within the quasi-planar substrate110, wherein the one or more laterally-mobile effector molecule types120, 130, 140 is configured to responsively interact with one or morecell types 150, and at least one of the one or more laterally-mobileeffector molecule types 120, 130, 140 is configured to interactsequentially with the one or more cell types 150. The effector moleculetype can be a ligand molecule 120. The effector molecule type can be areceptor molecule 130. The effector molecule type can be a cytokine 140.The device can further include one or more sensors 160 configured todetect the at least one of the one or more laterally-mobile effectormolecule types 120, 130, 140 interacting with the one or more cell types150; and a controller 170 in communication with the one or more sensors160, wherein the controller 170 is configured to responsively initiatemodification or movement of the one or more laterally-mobile effectormolecule types 120, 130, 140 and the one or more cell types 150 in thequasi-planar substrate 110.

Referring to FIG. 1A, depicted is a partial diagrammatic view of anaspect of an embodiment of a device 100 including a quasi-planarsubstrate 110; and one or more laterally-mobile effector molecule types120, 130, 140 embedded within the quasi-planar substrate 110, whereinthe one or more laterally-mobile effector molecule types, e.g., a ligandmolecule 120, is configured to responsively interact with one or morecell types 150.

Referring to FIG. 1B, depicted is a partial diagrammatic view of anaspect of an embodiment of a device 100 including a quasi-planarsubstrate 110; and one or more laterally-mobile effector molecule types120, 130, 140 embedded within the quasi-planar substrate 110, whereinthe one or more laterally-mobile effector molecule types, e.g., areceptor molecule 130, is configured to responsively interact with theone or more cell types 150. The one or more sensors can sense aninteraction between the ligand molecule 120 and the one or more celltypes 150. The controller 170 in communication with the one or moresensors 160 is configured to responsively initiate modification ormovement of the ligand molecule 120 and the one or more cell types 150in the quasi-planar substrate 110 such that the one or more cell types150 can interact with a second laterally-mobile effector molecule types,e.g., a receptor molecule 130.

Referring to FIG. 1C, depicted is a partial diagrammatic view of anaspect of an embodiment of a device 100 including a quasi-planarsubstrate 110; and one or more laterally-mobile effector molecule types120, 130, 140 embedded within the quasi-planar substrate 110, whereinthe one or more laterally-mobile effector molecule types, e.g., acytokine 140, is configured to responsively interact with the one ormore cell types 150. The one or more sensors can sense an interactionbetween the receptor molecule 130 and the one or more cell types 150.The controller 170 in communication with the one or more sensors 160 isconfigured to responsively initiate modification or movement of thereceptor molecule 130 and the one or more cell types 150 in thequasi-planar substrate 110 such that the one or more cell types 150 caninteract with a third laterally-mobile effector molecule types, e.g., acytokine molecule 140. The one or more cell types 150 interact with thecytokine molecule 140 and are subsequently released from the interactionwith the cytokine molecule 140.

Referring to FIG. 2, depicted is a partial diagrammatic view of oneaspect of an embodiment of a method 200 including embedding 210 one ormore laterally-mobile effector molecule types at least partially withina quasi-planar substrate of a device, transferring 220 one or more celltypes to the quasi-planar substrate of the device, interacting at leastone of the one or more laterally-mobile effector molecule types with theone or more cell types, sensing 230 at least one aspect of aninteraction between the one or more laterally-mobile effector moleculetypes and the one or more cell types, and controlling 240, in responseto the sensing, initiation of modification of at least one of the one ormore laterally-mobile effector molecule types, the quasi-planarsubstrate, and the one or more cell types.

The device can include one or more laterally-mobile effector moleculetypes at least partially embedded within the quasi-planar substrate thatare activating receptors for T_(H) cells that comprise an immunesynapse, including for example, a viral peptide antigen complexed withMHC class II; costimulatory molecules, e.g., CD4, CD80 or CD86; andintegrins, e.g., ICAM-1. The device can act as an immune synapse tostimulate T cells. See e.g., Doh and Irvine, Proc. Natl. Acad. Sci. USA103: 5700-5705, 2006, which is incorporated herein by reference. Thedevice can further deliver one or more laterally-mobile effectormolecule types at least partially embedded within the quasi-planarsubstrate that include cytokines, e.g., IL-2, IL-12, or IFN-γ, to theT_(H) cell in response to signals from sensors in the device indicatingthat a T_(H)1 cell has bound to activating receptors such as thosedescribed above which comprise an immune synapse. Alternatively,autoreactive T cells or pathogenic T cells can be selectivelyinactivated or killed by processing in the device. For example,autoreactive CD8⁺ T cells can be selectively engaged by MHC class I plusan autoantigen peptide, e.g., a peptide from myelin basic protein (MBP).MHC I plus MBP-peptide complex can be presented on the surface of thephospholipid bilayer of the device and bind autoreactive T cells. T cellbinding to the MHC plus MBP-peptide complex can activate a sensor (e.g.,a surface plasmon resonance sensor that initiates an electrical signalto a controller. The controller can respond by inducing apoptosis in theCD8⁺ T cell. The controller can actuate valves and pumps to deliveranti-Fas receptor (FasR, CD95) antibody to induce apoptosis in theengaged T cell. Alternatively, the controller can respond by presentingthe membrane protein, Fas ligand (FasL, CD178) on the surface of thebilayer to engage FasR and induce apoptosis in the CD8⁺ T cell. See,e.g., Holler et al., Mol. Cell. Biol. 23: 1428-1440, 2003 which isincorporated herein by reference.

The device as described herein can be used to transfect, grow, induce,activate, de-differentiate or differentiate stem cells for applicationsin regenerative medicine. Stem cells including, but not limited to,hematopoietic stem cells, myeloid stem cells, mesenchymal stem cells,embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs)can be used for transplantation to treat autoimmune diseases, cancer,and many other diseases. For example the device can be used to reprogram(i.e. de-differentiate) adult human fibroblasts to become iPSCs bymicroinjecting the transcription factors: October 4, Sox 2, Klf 4 andMyc. See e.g., Graf and Enver, “Forcing cells to change lineages.”Nature 462: 587-594, 2009 which is incorporated herein by reference.Alternatively, stem cells can be differentiated and/or cultivated in thedevice to generate specific cell lineages. The specific cell lineages,neuronal cells, or hematopoietic cells can be used to treat patientswith neurodegenerative diseases or hematological cancers, respectively.The device can be used to differentiate ESC to become neuroepithelialcells and then dopamine (DA) neurons. See e.g., Yan et al., “Directeddifferentiation of dopaminergic neuronal subtypes from human embryonicstem cells.” Stem Cells 23: 781-790, 2005 which is incorporated hereinby reference. ESCs can be grown in the device in a neural growth mediacontaining fibroblast growth factor-2 to generate neural epithelialcells. The neural epithelial cells can then be moved to a cell baycontaining a laminin substrate and treated with sonic hedgehog andfibroblast growth factor-8 to generate DA neurons. The device can detectDA released into the media by DA neurons and also sense the expressionof neuron-specific marker proteins such as synaptophysin and c-Retindicating functional DA neurons have differentiated. A controller canrespond to signals from the neuronal cell sensors by transporting thedifferentiated DA neurons to a collection compartment where they can beharvested for transplantation to Parkinson's disease patients whose DAneurons have degenerated.

The device can also be used to obtain hematopoietic progenitor cellsthat can be used for transplantation into leukemia and lymphoma patientsto regenerate the hematopoietic systems of the patients. To obtainautologous, i.e., “matched” stem cells the device can be used to produceiPSCs by reprogramming a patient's fibroblasts as described above. Seee.g., Graf and Enver, Ibid. and Choi et al., “Hematopoietic andendothelial differentiation of human induced pluripotent stem cells.”Stem Cells 27: 559-567, 2009, which are incorporated herein byreference.

The iPSCs are differentiated to hematopoietic progenitor cells by growthin the device over a feeder layer of bone marrow stromal cells, e.g.,OP9 cells. See Vodyanik et al., Blood 105: 617-626, 2005 which isincorporated herein by reference. After culture for approximately 8 daysthe device can detect and collect hematopoietic progenitors (CD34⁺CD43⁺cells; see e.g., Choi et al., Ibid.) which can be used to repopulatemultiple hematopoietic lineages in leukemia and lymphoma patientsundergoing bone marrow transplantation.

The device as described herein can be used to cultivate and fertilizemammalian oocytes prior to implantation. Oocytes can be retrieved bytransvaginal aspiration and cultured in the device with human tubalfluid medium at 37° C. For fertilization, approximately 10⁵ motile spermcan be injected into a cell bay containing an oocyte, and fertilizationis allowed to proceed for approximately 16-20 hours. See e.g., Rossatoet al., Hum. Reprod. 14: 694-697, 1999 which is incorporated herein byreference. The device can detect pronuclei in fertilized oocytesmicroscopically and with optical sensors. After 46-48 hours the devicecan also detect cell cleavage and evaluate embryo quality. Opticalsensors in the device signal a controller when high quality embryos aredetected. The controller can actuate microfluidics including micropumpsand valves in the device to transport and collect embryos forimplantation.

The device as described herein can be constructed with a quasiplanarsubstrate including a solid substrate supporting a fluid substrate thatincludes laterally-mobile ligand molecules and sensors. The ligands andsensors can detect and engage cells in the device and signal to acontroller. The solid substrate can be comprised of silica, glass,polydimethylsiloxane, plastic, metal, fiber, or membrane. A micropatternarray on the solid substrate can be formed using microfabricationtechniques such as photolithography, acid etching, soft lithography, ordrilling. For instance, micromachining techniques include filmdeposition processes, such as sputtering, spin coating in combinationwith chemical vapor deposition, laser fabrication, photolithographictechniques, or etching methods. Etching can be performed by wetchemical, plasma, reactive ion or sputtering processes. Micromachiningmethods are described, for example, in Petersen, Proceedings of the IEEE70: 420-457, 1982, which is incorporated herein by reference. Methodscan be used to construct microfluidic channels, cell bays, on-chippumps, piezoelectric actuators, optical tweezers, valves and reservoirsin microchips. See, e.g., U.S. Pat. No. 7,435,578 issued to Wikswo etal. on Oct. 14, 2008 and Enger et al., Lab Chip 4: 196-200, 2004, whichare incorporated herein by reference. For example, the microfluidicslayer can be fusion bonded to a silicon layer containing sensing andcontrol elements. The microfluidics layer can include channels, cellchambers and silicon containers with submicron holes. The channels andcontainers are etched into the substrate by reactive ion etching of asilicon nitride mask and a non-isotropic potassium hydroxide (KOH) etch.Alternatively, the micropattern array device can be constructed using asoft lithography process. See e.g., Liu et al., Lab Chip 9: 1200-1205,2009 which is incorporated herein by reference. A microfluidic devicecan be constructed from polydimethylsiloxane (PDMS) using aliquid-molding procedure to create interconnected channels, single cellbays, reservoirs, valves, gates and cell collection traps.Photolithography can be used to fabricate glycerol molds that can beused to cast PDMS microchips. The microchips can be drilled to createaccess holes, or to install pumps, valves or gates. The device caninclude phospholipid bilayers, ligands, sensors and electrodes added tothe cell bays. The microfluidics can be enclosed by bonding the PDMSmicrochip to a glass slide. Channels with a width ranging fromapproximately 50 μm to 500 μm and an apical height ranging fromapproximately 10 μm to 50 μm can be created to connect cell chambers,reservoirs, cell collection traps and external ports. Multiple parallelmicrofluidic circuits can be created in a 25 mm×75 mm microchip. Seee.g., Liu et al., Ibid. The device can include on-chip pumps constructedfrom a reservoir covered with a flexible membrane. The flexible membranecan be moved in or out by changing the length of a piezoelectric elementto pump fluid through the reservoir. An array of individuallyaddressable piezoelectric filaments can be utilized to provide separateactuation of multiple pumps. The piezoelectric filaments can alsoprovide valving by pinching closed sections of the channels between thereservoirs. The device can also include an optical tweezer system tomanipulate cells within the microfluidic channels and cell chambers.Optical tweezers combined with microfluidic chips have been described.See e.g., MacDonald et al., Nature 426: 421-424, 2003 and Enger et al.,Ibid., which are incorporated herein by reference. Optical tweezers canbe constructed by directing a laser beam through the objective of amicroscope. For example, an optical tweezer can be constructed from anArgon ion pumped titanium-sapphire laser (with a wavelength range of 780to 850 nm) and a Nikon microscope with a Nikon IR 100× objective with anumerical aperture of 1.3. Individual cells are trapped in the focalpoint of the laser beam and they can be moved between channels orchambers of a microfluidic device by changing the direction of the laserbeam. Alternatively, cells can be sorted in a microfluidic device withan optical tweezer that differentially diverts the flow path of selectedcells.

The device can include cell bays ranging in size between approximately20 μm and 500 μm in diameter can be designed to hold one cell ormultiple cells. Channels approximately 10 μm to 200 μm in width canconnect the entry ports, cell bays, exit ports and cell traps to createa microfluidic circuit on the device. The quasi-planar substrateincludes a fluid or semi-fluid substrate layered over a solid substrate,and the fluid or semi-fluid substrate can contain one or morelaterally-mobile ligand molecules and sensors that can move laterally inthe plane of the substrate. The fluid substrate can be a membrane, e.g.,a phospholipid bilayer, a phospholipid monolayer, or a liquid substratethat is immiscible with the fluids above and below it. Phospholipidbilayers can be comprised of lipids such as phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, cholesterol andsphingomyelin. For example, phospholipid vesicles can be prepared fromcholesterol and L-α-phosphatidylcholine. See, e.g., U.S. PatentApplication No. 2005/0208120 published on Sep. 22, 2005 which isincorporated herein by reference. Phospholipid vesicles can be appliedto phospholipid bilayer-compatible regions of the device, for example,within cell chambers where they can form a planar phospholipid bilayersurround by fluid layers, i.e., aqueous layers, above and below. Seee.g., U.S. Pat. No. 6,228,326 issued to Boxer et al. on May 8, 2001,which is incorporated herein by reference. Phospholipid bilayer vesiclescan contain ligands and receptors at least partially embedded in theplanar bilayer when the vesicles are applied to a bilayer-compatiblesurface.

Laterally-mobile ligand molecules and sensors can be embedded in orattached to the fluid or semi-fluid substrate and oriented to bindsurface molecules present on the cells of interest above thequasi-planar substrate and to signal via the media below. Ligands caninclude, but are not limited to, receptor proteins, antibodies, integralmembrane proteins, glycophosphatidylinositol-anchored proteins, fusionproteins, conjugated proteins, aptamers or polymers. Sensors can befixed in place and surrounded by a fluid substrate. Sensors attached tometal electrodes can be layered onto the solid substrate of the device.See e.g., U.S. Pat. No. 6,228,326, Ibid. which is incorporated herein byreference. Sensors can be attached to a metal electrode usingalkanethiol linkers. See e.g., Savran et al., Analytical Chemistry 76:3194-3198, 2004, which is incorporated herein by reference. Sensorrefers to any device that can detect a measurable quantity. For example,a sensor can be a thermal detector, an electrical detector, a chemicaldetector, an optical detector, an ion detector, a biological detector, aradioisotope detector, an electrochemical detector, a radiationdetector, an acoustic detector, a magnetic detector, a capacitivedetector, a pressure detector, an ultrasonic detector, an infrareddetector, a microwave motion detector, a radar detector, an electriceye, an image sensor, or a combination thereof. Sensors can use opticaldetection methods including ellipsometry (see, e.g., Corsel, et al. J.Colloid Interface Sci 111: 544-554, 1986, which is incorporated hereinby reference), optical wave guidance (see, e.g., Nellen and Lukosz,Sensors and Actuators B 1: 592-596, 1990, which is incorporated hereinby reference) or surface plasmon resonance (see, e.g., Cullen, et al.Biosensors 3: 211-225, 1988, which is incorporated herein by reference).Surface plasmon resonance is advantageous for monitoring molecularinteractions in real-time, enabling a sensitive and comprehensiveanalysis of the degree of binding interactions between two proteins. Ona quasi-planar substrate, a support grid can be fabricated by making anarray of conductive regions, e.g., gold, separated by lipid bilayerbarrier regions. Biosensors can contain conductive regions,bilayer-compatible regions or bilayer barrier regions, i.e., regions towhich lipid bilayers do not adhere. See e.g., U.S. Pat. No. 6,228,326Ibid., which is incorporated herein by reference. Examples of barriermaterials include, but are not limited to: certain polymers, e.g.,positive photoresist, metals, e.g., gold, and minerals, e.g., aluminumoxide. A very thin polymer film, such as polyacrylamide or dextran, canbe deposited on the conductive regions, e.g., gold, to formbilayer-compatible surface regions. The polyacrylamide can be coupled toa surface by 3-methacryl-oxypropyl-trimethoxy-silane (MPTS; Serva,Heidelberg, Germany). See e.g., Kuhner, et al., Biophysical Journal 67:217-226, 1994, which is incorporated herein by reference. Phospholipidbilayers containing ligand molecules and sensors can be deposited onbilayer-compatible regions as described above. See e.g., U.S. Pat. No.6,228,326 Ibid., which is incorporated herein by reference. The lipidbilayer-compatible region is located in a cell bay of the device where asolution including cells of interest can be passed over the surfacecontaining the array of receptor-studded lipid expanses. The grid isilluminated at an angle with a light emitting diode (LED), and reflectedlight is analyzed with a photodetector. An evanescent electric field canbe generated by the interaction of incident light with the conductivegold layer. Reflected light from the interaction is sensitive to theenvironment of a layer extending about 1 μm (approximately 760 nm) fromthe receptors into the medium. Changes in the environment of thereceptor, such as changes caused by binding of a ligand to the receptor,are detected as changes in the reflectance intensity at a specific angleof reflection, termed the resonance angle.

Various cell conditions or cell interactions can be detected on thedevice by sensors using capacitative detection or impedance analysis.Cell conditions or cell interactions include, but are not limited to,cell engagement, cell binding, cell bay occupancy, ligand binding to areceptor, protein expression, cell product secretion or cell analytes.Cell conditions or cell interactions can be detected by sensors.Capacitative detection or impedance analysis utilizes an electrodeincorporated into the bilayer-compatible surface region, and a “ground”electrode placed in the aqueous phase below the bilayer. A voltage froma variable-frequency function generator is used to generate a selectedvoltage waveform which is fed across selected array elements. Thepeak-to-peak amplitude of the voltage is typically on the order of about10 V, but can be substantially less. The voltage is applied over a rangeof frequencies and the capacitance is determined from the measuredcurrent as a function of signal frequency using standard signalprocessing techniques. The application of capacitance measurements andimpedance analyses of supported bilayers are discussed, for example, inStelzle et al., Biochim. Biophys. Acta. 981: 135-142, 1989 which isincorporated herein by reference. Sensor electrodes can be constructedon glass plates onto which chromium layers of approximately 20-50 nmthickness are deposited. The electrode surface can be made hydrophilicby argon sputtering and lipid bilayers can be deposited on the electrodesurface and covered by an aqueous solution. Measurement of specificcapacitance, specific resistance and electrolyte resistance across thelipid bilayer can be used to detect protein adsorption to the bilayerfrom the aqueous phase. Cell proteins such as cytokines can be detectedby electrodes with lipid bilayers containing specific receptors for thecytokines. Also aptamers can be used as sensors to detect cell surfaceproteins and cell products such as cytokines. Aptamers are small RNA orDNA molecules that specifically bind small molecules, proteins,carbohydrates, and lipids with high specificity and high affinity.Aptamers specific for cell surface antigens and/or cell products can beattached to gold electrodes constructed in a cell chamber usingthiol-modified aptamers in solution to react with the gold surface. Seee.g., U.S. Pat. No. 6,228,326, Ibid and Savran et al., AnalyticalChemistry 76: 3194-3198, 2004, which are incorporated by referenceherein. Binding of cell surface proteins or cell products to thecorresponding aptamers on sensors can transduce an electrical signalthat is received by the controller of the device. Cell engagement, cellbinding, cell bay occupancy, protein expression, cell activation, cellfertilization, cell product secretion and cell analytes can be detectedby sensors and signaled electronically to a controller that can respondto the signals.

Controller refers to any device that can receive, process and presentinformation. For example, a controller can be, e.g., one microprocessor,several microprocessors coupled together, a computer, or severalcomputers coupled together. The controller can have multiple addressescorresponding to each of the sensors in the device. The controller canbe programmed to respond appropriately to each of the sensors. Forexample, a cell engaging a sensor in cell bay 1 can trigger a signal tothe controller. The controller, in turn, can respond by opening a valveand actuating a micropump to inject a cytokine into cell bay 1 and byactuating the presentation of ligands and receptors in cell bay 1designed to interact with the cell. The controller can also respond bycausing microfluidic flow in the cell bay to wash the cell or to movethe cell to a new location in a second cell bay. By closing and openingpiezoelectric gates the device can control a path that the cell takesthrough the device. The controller can also respond by delivering anelectric charge to cell bay 1 to electroporate the cell and to injectone or more gene expression constructs into the cell. The controller canalso respond to sensors in cell bay 1 by providing cytokines, ligands,receptors, microfluidic flow or other modifiers to cell bay 2. Forexample, sensors can detect the activation of a T cell, e.g. bydetection of IL-2 production, in cell bay 1 and signal to the controllerwhich can respond by modifying cell bay 2. To capture activated T cellsin cell bay 2, the controller can respond by providing membrane-boundantibodies that recognize T cell activation antigens, e.g., anti-HLA-DRand anti-IL-2 receptor a (CD25) in cell bay 2. Phospholipid bilayervesicles containing membrane bound antibodies (anti-HLA-DR andanti-CD25) can be injected into cell bay 2 to provide a planar lipidbilayer that can capture activated T cells. The controller also canrespond by injecting cytokines, e.g., IL-21 and IL-15 into cell bay 2 topromote the survival and maturation of the T cell. See, e.g., Cotner etal., J. Exp. Med. 157: 461-472, 1983 and Zeng et al., J. Exp. Med. 201:139-148, 2005 which are incorporated herein by reference.

PROPHETIC EXAMPLES Example 1 A micropattern Array Device toDifferentiate, Activate and Collect Regulatory T Cells

An ex vivo device is constructed to responsively interact withregulatory T (Treg) cells from a patient having autoimmune disease. Thedevice includes a lipid bilayer on a quasi-planar silicon wafersubstrate and laterally mobile ligands embedded within the lipid bilayerthat interact with receptors on the Treg cells. The device also includesoptical sensors and optical waveguide sensors, and a controller thatresponds to the sensors by delivering effector molecules, e.g.,cytokines and receptors to the T cells. The device is constructed usinga combination of microelectronic and microfluidic methods to containsingle cell chambers connected by channels with gates and collectionchambers. The chambers and channels are comprised of multiple layersthat include a solid support (silica), an aqueous fluid layer for signaltransduction, a lipid bilayer, and a cell fluid media. Treg cells enterthe device at an entry port and traverse a microfluidic circuit withinthe device that activates and collects Treg cells from the patient. Theactivated Treg cells suppress autoreactive T cells to regulate anautoimmune response to self-antigens in autoimmune disease in thepatient.

A device including a micropatterned array is constructed usingmicrofabrication methods to contain single cell compartments composed ofmultiple layers that include ligands, sensors and controllers thatinteract responsively to detect, activate and collect Treg cells.Methods are used to create micropattern arrays of lipid bilayerscontaining membrane proteins. See e.g., U.S. Patent Application No.2008/0317724 entitled “Micropatterned T Cell Stimulation” published onDec. 25, 2008 and U.S. Pat. No. 6,228,326 issued to Boxer et al. on May8, 2001, which are incorporated herein by reference. A patterned supportgrid is constructed from 100 mm diameter silicon wafers (available fromSilrec Corp., San Jose, Calif.). The wafers are maintained at 1000° C.in an oxidation furnace to generate a layer of silicon oxide.

Positive photoresist (S-1800 available from Shipley Inc., Marlborough,Mass.) is spin-coated onto the wafers using a track coater (SiliconValley Group, San Jose, Calif.), and then the wafers are exposed to UVlight at approximately 10 mW/cm² through a photolithographic mask. Themicropattern is developed with tetramethylammonium hydroxide developer(Shipley Inc., Marlborough, Mass.), and the wafers are etched forapproximately three minutes in argon plasma. The micropattern isdesigned with single cell chambers approximately 20 μm in diameterconnected by channels approximately 10 μm in width that delineate amicrofluidic circuit. Single cell chambers are coated with an aqueouslayer and a lipid bilayer.

Continuous planar lipid bilayers are formed on the micropattern arrayusing a suspension of phospholipid vesicles containing integral membraneproteins. Phospholipid vesicles are constructed using lipid raftscontaining membrane ligands (antibodies or antibody fragments) withspecificity for Treg surface markers and activation antigens, CD4, CD25and CD3.

Antibodies or antibody fragments that act as membrane ligands torecognize Treg surface markers are expressed using recombinant DNAmethods in Chinese hamster ovary (CHO) cells. Anti-CD4, anti-CD25 andanti-CD3 membrane proteins are isolated in lipid rafts from the CHOcells expressing the recombinant antibodies or antibody fragments.Recombinant antibodies or antibody fragments that act as membraneligands are constructed from single chain variable fragments (scFv)fused to a transmembrane domain. Recombinant human antibodies are selectfrom phage display scFv libraries. See e.g., Pansri et al., BMCBiotechnology 9(6): 2009; doi: 10.1186/1472-6750-9-6 which isincorporated herein by reference. scFv antibody fragments that recognizeCD4, CD25 and CD3 are isolated and engineered to contain a transmembranedomain at the carboxy terminus of each scFv protein. The scFv proteinsare constructed as tandem repeats of each scFv. See e.g., Herrmann etal., Cancer Res. 68: 1221-1227, 2008, which is incorporated herein byreference. DNA sequences of immunoglobulin are constructed encoding aspacer region, transmembrane domain, and cytoplasmic region frommembrane IgM (mIgM). See, e.g., Michnoff et al., J. Biol. Chem. 269:24237-24244, 1994, which is incorporated herein by reference.Complementary DNA (cDNA) encoding scFv specific for CD4, CD25 and CD3are obtained by molecular cloning using the bacteriophage clonesselected above. Methods to isolate mRNA, clone cDNA and determine DNAsequences are provided. See e.g., U.S. Pat. No. 7,141,656 entitled “MHCComplexes and Uses Thereof” issued to Rhodes et al. on Nov. 28, 2006 andSambrook and Russell, “Molecular Cloning: A Laboratory Manual”, ThirdEdition, 2001, Cold Spring Harbor Laboratory Press, Woodbury, N.Y.,which are incorporated herein by reference. A DNA sequence encoding thespacer, transmembrane domain and cytoplasmic tail of mIgM (GenbankAccession No.: AAB59651; amino acids No. 438-475) is fused to the 3′ endof each scFv cDNA. The anti-CD3 scFv-mIgM fusion gene and the anti-CD25scFv-mIgM fusion gene are inserted in a bicistronic mammalian cellexpression vector. See e.g., Product Information Sheet: “pIRES Vector”available from Clontech Laboratories, Inc., Mountain View, Calif. whichis incorporated herein by reference. A second mammalian cell expressionvector encoding the anti-CD4 scFv-mIgM fusion gene is constructed withan alternate selectable marker, e.g., dihydrofolate reductase (DHFR) toallow coselection of the scFv vectors with methotrexate and G418. CHOcells are transfected with one or both scFv-mIgM expression vectorsusing Lipofectamine™ (available with protocols from Life TechnologiesCorp., Carlsbad, Calif.). Stable clones are selected for resistance toG418, or methotrexate, or to both drugs. To test for the expression ofthe scFv-mIgM proteins the cells are stained with fluorescent antibodies(e.g., anti-kappa variable region (V_(k)) antibodies) and analyzed on aflow cytometer. Antibodies, reagents, protocols and flow cytometers areavailable from BD Biosciences, San Jose, Calif. Stable CHO cell linesexpressing anti-CD4 and anti-CD25 and anti-CD3 on the cell surface areexpanded in a bioreactor to provide a source of lipid rafts containinganti-CD4, anti-CD25 and anti-CD3 scFv membrane proteins.

To isolate lipid rafts from mammalian cell lines includingdetergent-free lipid rafts, a carbonate step gradient method is used.See, e.g., Macdonald et al., J. Lipid Research 46: 1061-1067, 2005 whichis incorporated herein by reference. The cells are washed and scrapedinto a 500 mM sodium carbonate buffer, pH 11.0, containing 7 differentprotease inhibitors. The cells are lysed using a Dounce homogenizer anda syringe with a 23 gauge needle and a Branson Sonifier 250. The cellhomogenate is fractionated on an OptiPrep™ gradient (available fromAxis-Shield PoC, Oslo, Norway) and fractions containing lipid rafts areidentified by immunoblotting with antibodies specific for membraneproteins (e.g., anti-CD4, anti-CD25 and anti-CD3 scFv).

Phospholipid vesicles are prepared from cholesterol andL-α-phosphatidylcholine. See, e.g., U.S. Patent Application No.2005/0208120 published on Sep. 22, 2005, which is incorporated herein byreference. Cholesterol and L-α-phosphatidyl choline are combined at amolar ratio of 2:7 in chloroform. The chloroform is evaporated awayusing an argon stream. The vesicles are resuspended in a 140 mM NaCl, 10mM Tris HCl, 0.5% deoxycholate at pH 8 and sonicated for three minutes.Lipid rafts containing scFv specific for Treg markers (e.g., CD4, CD25,and CD3) are inserted in the vesicles by combining the lipid raftscontaining anti-CD4, anti-CD25 and anti-CD3 scFv with the phospholipidvesicles at a 1:10 molar ratio and dialyzing for 72 hours at 4° C.versus phosphate-buffered saline. The vesicles are characterized toassess vesicle size and the amount of anti-CD4, anti-CD25 and anti-CD3scFv protein incorporated in the vesicles. Vesicle size is determinedusing dynamic light scattering and flow cytometry (see e.g., U.S. PatentApplication No. 2005/0208120, Ibid.). For example, vesicles with a meandiameter of approximately 25 nanometers are optimal. To quantify scFvprotein on the vesicles, the vesicles are analyzed on a flow cytometerafter staining with FITC-labeled anti-V_(k) antibody. Vesicles aresorted based on FITC fluorescence, forward scatter and side scatter toisolate and count vesicles with scFv. Surface scFv protein on thevesicles is quantified using an enzyme-linked immunosorbent assay(ELISA). Methods to analyze vesicles by flow cytometry and to quantifyscFv and other proteins by ELISA are provided. See e.g., U.S. PatentApplication No. 2005/0208120, which is incorporated herein by reference.

Phospholipid bilayers are formed on the micropattern arrays bycontacting the patterned surface of the wafer support grids with asuspension, prepared as described above, containing approximately 25 nmdiameter vesicles. Vesicles in the suspension spontaneously assemble toform a continuous single bilayer on the bilayer-compatible regions ofthe support grid. Regions of the grid where photoresist remains are notbilayer compatible and form barrier regions or boundaries around thelipid bilayer. Methods to construct bilayer compatible regions andbarrier regions are provided. See, e.g., U.S. Pat. No. 6,228,326, whichis incorporated herein by reference.

To construct a differentiation and activation path for Treg cells, twosingle cell compartments (bays) connected by microfluidic channels arecoated with vesicles containing different scFv. Cell bay 1 is coatedwith anti-CD4 scFv vesicles; cell bay 2 is coated with anti-CD25 plusanti-CD3 vesicles. A channel exits from cell bay 2 and connects to acell collection trap where activated Treg cells are recovered.Microfluidic cell traps are provided. See e.g., Liu et al., Lab Chip 9:1200-1205, 2009, which is incorporated herein by reference. Methods toconstruct microfluidic channels, on-chip pumps, piezoelectric actuators,valves and reservoirs in microchips are described. See e.g., U.S. Pat.No. 7,435,578 issued to Wikswo et al. on Oct. 14, 2008 which isincorporated herein by reference. Microchips are constructed to maintaincontrolled temperature (approximately 37° C.) and 5% CO₂ atmosphere topromote mammalian cell growth.

The device including the micropatterned array is constructed withoptical sensors located underneath the lipid bilayer coating the singlecell bays. Methods to construct microchips with optical sensors areprovided. See, e.g., Nellen and Lukosz, Sensors and Actuators B1:592-596, 1990; U.S. Pat. No. 7,435,578 Ibid. and U.S. Patent ApplicationNo. 2004/0219605 by Finkel et al. published on Nov. 4, 2004, which areincorporated herein by reference. The device incorporates afiber-coupled optical system that includes an optical waveguide sensor(available from MicroVacuum Ltd., Budapest, Hungary; see e.g., OWLSApplication Notes NO-004, “Quantification of Cell Adhesivity” which isincorporated herein by reference). A laser light source is integratedinto the cell bay to irradiate the lipid bilayer and the waveguidesensor. Optical signals are conveyed through the waveguide to attachedoptical fibers and finally to a potentiometric light detector. Forexample, a CD4-positive T cell binds to anti-CD4 scFv presented on thesurface of bay 1 and alters the optical signal emanating from thewaveguide sensor. The altered optical signal is detected by thepotentiometric light detector and relayed to a controller where aresponse is initiated. Optical sensors immediately below the lipidbilayers in the cell bays containing anti-CD4, anti-CD25, and anti-CD3scFv detect when a cell engages, i.e., binds, the scFv and occupies orleaves the cell bay. The controller contains microcircuitry and programsto respond when cells are located in bay 1 or bay 2. For example, CD4+ Tcells detected in bay 1 are provided with transforming growth factor β(TGF-β) to promote the development of Treg cells. See e.g., Bettelli etal., Nature 441: 235-238, 2006 which is incorporated herein byreference.

After CD4⁺ T cells are exposed to human TGF-β1 (at approximately 3ng/mL) for approximately 3 days, the treated cells are moved by pumpingfluid via a channel to bay 2 for activation. Individual Treg cellsexpressing CD25 and CD3 are captured in cell bay 2 by anti-CD25 andanti-CD3 scFv recognizing each antigen. The CD25⁺, CD3⁺Treg cells aredetected by optical sensors as described above. Controllers respond tothe CD25⁺, CD3⁺Treg cells docked in bay 2 by provision of interleukin 2(IL-2) at 20 IU/mL to the cells. The IL-2-treated T cells are incubatedapproximately 72 hours. The differentiated and activated Treg cells aremoved by fluid flow to a cell collection trap to be harvested or infuseddirectly into the patient to treat autoimmune disease.

Example 2 A Method to Treat A Patient with Autoimmune Disease by Ex VivoLeukapheresis and Activation and Collection and Reinfusion of AntigenSpecific (Autoreactive Suppressors) Treg Cells

A patient with relapsing-remitting multiple sclerosis (MS) is treatedwith an ex vivo device that differentiates, activates and collectsregulatory T (Treg) cells for immune cell therapy. Treg cells suppressautoreactive T cells associated with multiple sclerosis in a patient.See e.g., Yu et al., J. Immunol. 174: 6772-6780, 2005 which isincorporated herein by reference. The activated Treg cells are reinfusedinto the patient to reduce or eliminate the autoimmune attack on thecentral nervous system associated with MS. T cells are obtained from theperipheral blood of the MS patient, and potential Treg cells areisolated using leukapheresis methods. See e.g., Hoffmann et al., Blood104: 895-903, 2004 which is incorporated herein by reference. Treg cellsare processed in a micropattern array device that responsively interactswith the Treg cells to differentiate, activate and collect the cellsprior to reinfusion into the patient.

Potential Treg cells are isolated from a MS patient by usingimmune-magnetic beads and fluorescence activated cell sorting of theblood of the patient. Peripheral blood mononuclear cells (PBMNC) areisolated from the peripheral blood of the patient using a leukapheresisprocedure and density gradient separation over Ficoll/Hypaque (availablefrom Sigma-Aldrich Chem. Co., St. Louis, Mo.). The leukocytes arestained with a phycoerythrin (PE)-conjugated anti-CD25 antibody andanti-PE magnetic beads (available from Miltenyi Biotec, BergischGladbach, Germany). CD25⁺ cells are enriched with the use of aMidi-MACS™ cell separator system (Miltenyi Biotec). CD25⁺ cells arestained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4antibodies and sorted using a FACStar™ Plus flow cytometer (from BDBiosciences, San Jose, Calif.) to isolate a CD4⁺CD25^(hi) population ofpotential Treg cells (see e.g., Hoffmann et al., 2004, Ibid.). By FACSsorting, approximately 1.8% of PBMNC are recovered as CD4⁺CD25 cellswith a purity of 98%. Leukapheresis can yield 10¹⁰ cells from a singleprocedure thus yielding maximally about 1.8×10⁸ potential Treg cells fordifferentiation, activation, and infusion.

A device including a micropattern array with multiple parallel paths forTreg cells is used to differentiate and activate potential Treg cells(i.e., CD4⁺CD25^(hi) cells). Each path or circuit includes two singlecell compartments (bays) coated with lipid bilayers containing differentantibodies, anti-CD4, anti-CD25, or anti-CD3 scFv antibodies, andconnected by microfluidic channels. Channel exits from cell bay 2connect to a cell collection trap where activated Treg cells arerecovered. Microfluidic cell traps are provided. See, e.g., Liu et al.,Lab Chip 9: 1200-1205, 2009 which is incorporated herein by reference.Cell bay 1 is coated with an anti-CD4 scFv in lipid bilayer membrane;cell bay 2 is coated with an anti-CD25 scFv plus anti-CD3 scFv in lipidbilayer membrane. Potential Treg cells suspended in media (e.g., RPMI1640 with 10% human serum albumin) enter the path through an entry valveand channel in the device and arrive, in succession at bay 1, at bay 2,and at the collection trap. The micropatterned array is also constructedwith optical sensors located underneath the lipid bilayer coating thesingle cell bays. The device incorporates a fiber-coupled optical systemthat includes an optical waveguide sensor, available from MicroVacuumLtd., Budapest, Hungary; see e.g., OWLS Application Notes NO-004,“Quantification of Cell Adhesivity” which is incorporated herein byreference. A laser light source is integrated into the cell bay toirradiate the lipid bilayer and the waveguide sensor. Optical signalsare conveyed through the waveguide to attached optical fibers and thento a potentiometric light detector. For example, a CD4⁺ T cell binds toanti-CD4 scFv presented on the surface of bay 1 and alters the opticalsignal emanating from the waveguide sensor. The altered optical signalis detected by the potentiometric light detector and relayed to acontroller where a response is initiated. Optical sensors immediatelybelow the lipid bilayers in the cell bays containing scFv (anti-CD4,anti-CD25 or anti-CD3) detect when a cell engages (i.e., binds) the scFvand occupies or leaves the cell bay. A controller containsmicrocircuitry and programs to respond to optical signals emanating froma specific path and single cell bay. For example, CD4⁺ T cells detectedin path 1, bay 1 are provided with transforming growth factor β (TGF-β)□to promote the development of Treg cells. See e.g., Bettelli et al.,Nature 441: 235-238, 2006 which is incorporated herein by reference.

After CD4⁺T cells are exposed to human TGF-β□ (at approximately 3 ng/mL)for approximately 3 days, the activated CD4⁺ T cells are moved to bay 2for activation by pumping fluid via a channel between bay 1 and bay 2.Individual Treg cells expressing CD25 and CD3 are captured in cell bay 2by anti-CD25 scFv and anti-CD3 scFv recognizing each antigen. The Tregcells are detected by optical sensors as described above. Controllersrespond to the CD25⁺, CD3⁺Treg cells docked in bay 2 are exposed tointerleukin 2 (IL-2) at 20 IU/mL and incubated approximately 72 hours.See e.g., Bollyky et al., J. Immunol. 183: 2232-2241, 2009 which isincorporated herein by reference. Finally the differentiated andactivated Treg cells are moved by fluid flow to a cell collection trapto be harvested or infused directly.

Treating a MS patient by reinfusion of activated, polyclonal, autologousTreg cells obtained from the micropattern device can suppressautoreactive T cells that are associated with the disease and reduce oreliminate the neurodegeneration associated with MS.

Example 3 A Device to Differentiate Peripheral Blood Dendritic PrecursorCells to Activated, Mature Dendritic Cells and Load them with ViralAntigens

A device including a micropattern array is constructed to differentiateand activate antigen presenting cells (APC) and load antigen onto theAPCs. The device is constructed as a microfluidic chip on a quasi-planarsubstrate having one or more laterally-mobile effector molecule types atleast partially embedded within the quasi-planar substrate, including afluid circuit comprised of interconnected channels and single cell baysthat responsively interact with the APC. Activated, antigen-loaded APCare used for immunotherapy and for immunology research.

The device including the micropattern array is constructed withchannels, single cell bays, sensors, controllers, ligands, antigens andcytokines that are incorporated in a microfluidic chip. Methods toconstruct microfluidic chips are provided. See, e.g., Liu et al., LabChip 9: 1200-1205, 2009 which is incorporated herein by reference. Amicrofluidic chip is constructed from polydimethylsiloxane (PDMS) usinga “liquid-molding” procedure to create interconnected channels, singlecell bays, reservoirs and cell collection traps for APC processing.Glass slides (25 mm×75 mm) are used to create wetting/dewettingpatterned surfaces. The slide is silanized (to render it hydrophobic)with 3-(trimethoxysilyl)propyl methacrylate (3% v/v) and baked at 120°C. for 4 hours, thus forming free methacrylate groups on the surface.Photolithography is used to fabricate micropatterns of hydrophilicregions (polymerized acrylamide) on the slide. The slide is dipped in apolar glycerol solution to create a micropattern of glycerol adheringonly to the hydrophilic regions. PDMS solution (Dow Corning, Midland,Mich.) is poured onto the glycerol micropattern to a depth ofapproximately 5 mm and cured at 60° C. for 1 hour. The PDMS substratewith a replicate channel structure is lifted off of the glycerol moldand drilled to create access holes, install pumps, valves and gates.Phospholipid bilayers, ligands, sensors and electrodes are added to thecell bays, and lastly the PDMS substrate is bonded onto a glass slidewith 1 minute treatment with oxygen plasma. Channels with a width ofapproximately 500 μm and an apical height of approximately 50 μm arecreated connecting cell chambers, reservoirs, cell collection traps andexternal ports. Approximately 75-150 parallel microfluidic circuits canbe created in a 25 mm×75 mm microchip (see e.g., Liu et al., Ibid.).Methods to construct microfluidic channels, on-chip pumps, piezoelectricactuators, valves and reservoirs in microchips are described (see e.g.,U.S. Pat. No. 7,435,578 issued to

Wikswo et al. on Oct. 14, 2008 which is incorporated herein byreference). Moreover methods to install gold electrodes and associatedcircuitry in microdevices are provided. See, e.g., U.S. Pat. No.6,228,326 issued to Boxer et al. on May 8, 2001 which is incorporatedherein by reference. The microdevice is constructed with heatingelements and thermocouple devices to maintain a controlled temperature(approximately 37° C.) to promote mammalian cell differentiation andsurvival. The microchip cell bays are coated with a quasi-planarsubstrate, a phospholipid bilayer that contains embedded ligands,receptors and sensors that responsively interact with the APC as theytravel a microfluidic circuit.

The device including the micropattern array contains multiple(approximately 75) parallel micro-circuits that engage, differentiate,activate and load dendritic cells (DC) with antigen. Methods to derivedendritic cells (DC) from peripheral blood mononuclear cells (PBMNC) areprovided. See, e.g., Krug et al., Eur. J. Immunol. 31: 3026-3027, 2001and Gilboa, J. Clin. Invest. 117: 1195-1203, 2007, which areincorporated herein by reference). Each micro-circuit includes an entrychannel leading to cell bay 1, an exit channel leading to cell bay 2,and an exit channel leading to a cell collection trap. Cell bay 1contains: surface-bound CD1c sensors; a granulocyte macrophage colonystimulating factor (GMCSF) injector; surface bound CD304 sensors; acytosine-guanosine (CpG)-rich oligodeoxynucleotide (ODN) injector; aninjector for a tumor associated antigen, prostate acid phosphatase(PAP); CD40 sensors; and CD80 sensors. Cell bay 2 contains: membranebound anti-CD54 (ICAM-1) scFv and anti-HLA-DR scFv. Cell bays areconstructed with a lipid bilayer containing embedded membrane proteinsand gold electrode regions bearing aptamer-based sensors. See e.g., Laiet al., Anal. Chem. 79: 229-233, 2007 which is incorporated herein byreference.

Membrane ligands that recognize dendritic cell surface markers areexpressed using recombinant DNA methods in Chinese hamster ovary (CHO)cells and isolated within lipid rafts. Some membrane ligands areconstructed from single chain variable fragments (scFv) fused to atransmembrane domain. Methods to select human antibody fragments fromphage display scFv libraries are provided. See, e.g., Pansri et al., BMCBiotechnology 9(6): 2009; doi: 10.1186/1472-6750-9-6, which isincorporated herein by reference. Single chain variable fragments thatrecognize CD54 and HLA-DR are isolated and engineered to contain atransmembrane domain at the carboxy terminus of each scFv protein. ThescFv proteins are constructed as tandem repeats of each scFv. See e.g.,Herrmann et al., Cancer Res. 68: 1221-1227, 2008 which is incorporatedherein by reference. DNA sequences of immunoglobulin (Ig) constructsencoding a spacer region, transmembrane domain, and cytoplasmic regionfrom membrane IgM (mIgM) are provided. See, e.g., Michnoff et al., J.Biol. Chem. 269: 24237-24244, 1994 which is incorporated herein byreference. Complementary DNA (cDNA) encoding anti-CD54 scFv andanti-HLA-DR scFv are obtained by molecular cloning using thebacteriophage clones selected from scFv libraries (see above). Methodsto clone genes and cDNA and to determine DNA sequences are provided.See, e.g., U.S. Pat. No. 7,141,656 entitled “MHC Complexes and UsesThereof” issued to Rhodes et al. on Nov. 28, 2006 and Sambrook andRussell, “Molecular Cloning: A Laboratory Manual”, Third Edition, 2001,Cold Spring Harbor Laboratory Press, Woodbury, N.Y., which areincorporated herein by reference. A DNA sequence encoding the spacer,transmembrane domain and cytoplasmic tail of mIgM (Genbank AccessionNo.: AAB59651; amino acids No. 438-475) is fused to the 3′ end of eachscFv cDNA. The anti-CD54 scFv-mIgM fusion gene and the anti-HLA-DRscFv-mIgM fusion gene are inserted in a bicistronic mammalian cellexpression vector. See, e.g., Product Information Sheet: “pIRES Vector”available from Clontech Laboratories, Inc., Mountain View, Calif. whichis incorporated herein by reference. Chinese hamster ovary (CHO) cellsare transfected with the expression vector encoding anti-CD54 scFv andthe expression vector encoding anti-HLA-DR scFv using Lipofectamine™transfection reagent (available with protocols from Life TechnologiesCorp., Carlsbad, Calif.). Stable clones are selected for theirresistance to G418. To test for the expression of the scFv-mIgMproteins, the cells are stained with fluorescent antibodies, e.g.,anti-kappa variable region (V_(k)) antibodies and analyzed on a flowcytometer. Antibodies, reagents, protocols and flow cytometers areavailable from BD Biosciences, San Jose, Calif.. Stable CHO cell linesexpressing anti-CD54 scFv and anti-HLA-DR scFv on their cell surfacesare expanded in a bioreactor to provide a source of lipid raftscontaining the corresponding membrane proteins.

Methods to isolate lipid rafts from mammalian cell lines are provided.See, e.g., Macdonald et al., J. Lipid Research 46: 1061-1067, 2005 whichis incorporated herein by reference. Detergent-free lipid rafts areprepared using a carbonate step gradient method. The cells are washedand scraped into a 500 mM sodium carbonate buffer, pH 11.0, containing 7different protease inhibitors. The cells are lysed using a Douncehomogenizer, a syringe with a 23 gauge needle, and a Branson Sonifier250. The cell homogenate is fractionated on an OptiPrep™ gradient(available from Axis-Shield PoC, Oslo, Norway) and fractions containinglipid rafts are identified by immunoblotting with antibodies specificfor membrane proteins (e.g., scFv, CD40 ligand; protocols and antibodiesfrom BD Biosciences, San Jose, Calif.).

Phospholipid vesicles are prepared from cholesterol andL-α-phosphatidylcholine. See, e.g., U.S. Patent Application No.2005/0208120 published on Sep. 22, 2005). Cholesterol andL-α-phosphatidyl choline are combined at a molar ratio of 2:7 inchloroform and the chloroform is evaporated away using an argon stream.The vesicles are resuspended in a 140 mM NaCl, 10 mM Tris HCl, 0.5%deoxycholate at pH 8 and sonicated for three minutes. Lipid raftscontaining membrane proteins (anti-CD54 scFv and anti-HLA-DR scFv)specific for dendritic cell activation receptors (e.g., CD54 and HLA-DR)are inserted in the vesicles by combining the lipid rafts containing themembrane proteins with the phospholipid vesicles at a 1:10 molar ratioand dialyzing for 72 hours at 4° C. versus phosphate-buffered saline.The vesicles are characterized to assess vesicle size and the amount ofmembrane protein incorporated in the vesicles. Vesicle size isdetermined using dynamic light scattering and flow cytometry (see e.g.,U.S. Patent Application No. 2005/0208120, Ibid.). For example, vesicleswith a mean diameter of approximately 25 nanometers are optimal. Toquantify scFv protein on the vesicles the vesicles are analyzed on aflow cytometer after staining with FITC-labeled anti-V_(k) antibody.Vesicles are sorted based on fluorescence, forward scatter and sidescatter to isolate and count vesicles presenting scFv. Surface scFvprotein on the vesicles is also quantified using an enzyme-linkedimmunosorbent assay (ELISA). Methods to analyze vesicles by flowcytometry and to quantify scFv and other proteins by ELISA are provided.See, e.g., U.S. Patent Application No. 2005/0208120, Ibid.

Phospholipid bilayers are formed on cell bay 1 and cell bay 2 bycontacting the PDMS microchip with a suspension, prepared as describedabove, containing approximately 25 nm diameter vesicles. Vesicles in thesuspension spontaneously assemble to form a continuous single lipidbilayer on the lipid bilayer-compatible regions of the microfluidicchips. Regions of the microchip that are not lipid bilayer compatible donot retain a lipid bilayer. Methods to construct lipid bilayercompatible regions and barrier regions are provided. See, e.g., U.S.Pat. No. 6,228,326 Ibid. CPG oligodeoxynucleotides are added to thephospholipid bilayer present in cell bay 1 to stimulate dendritic cellsvia their Toll-like receptors (TLR). CPG-ODN (available from InvivoGen,San Diego, Calif.) is condensed with a polycationic polymer,polyethyleneimine and injected onto the lipid bilayer coating bay 1.Positively charged, condensed CpG oligonucleotides bind to thenegatively charged phospholipid bilayer. See e.g., Ali et al., Sci.Transl. Med. 1: 8ra19, 2009 which is incorporated herein by reference.

The micropattern device also contains sensors to detect the engagementand activation of dendritic cells. Methods to make aptamer-based sensorsthat send an electronic signal are provided. See, e.g., Lai et al.,Anal. Chem. 79: 229-233, 2007 which is incorporated herein byreference). Aptamers are small RNA or DNA molecules that specificallybind small molecules, proteins, carbohydrates, and lipids with highspecificity and high affinity. Aptamers that recognize a specific targetare obtained by in vitro selection from a library ofoligodeoxynucleotides with random sequences (see Lee et al., Anal.Bioanal. Chem. 390, 1023-1032 (2008) which is incorporated herein byreference). Sensors to detect CD304, CD1c, CD40 or CD80 are constructedfrom aptamers selected to recognize recombinant CD304, CD1c, CD40 andCD80 protein (CD40 and CD80 proteins available from Abnova, Walnut, CA;CD304 protein available from R&D Systems, Minneapolis, Minn.). Aptamersspecific for CD304, CD1c, CD40 and CD80 are attached to gold electrodesconstructed in cell bay 1 (see e.g., U.S. Pat. No. 6,228,326, Ibid.)using thiol-modified aptamers in solution to react with the goldsurface. See e.g., U.S. Pat. No. 6,228,326, Ibid. For example, a DNAaptamer, 51 nucleotides long derivatized with a C6 thiol linker(HS(CH₂)₆PO₄) at its 5′ end can be immobilized on a gold-coated surfaceby incubation of a 10 μM aptamer solution with the gold surface forapproximately 20 minutes. See, Savran et al., Analytical Chemistry 76:3194-3198, 2004 which is incorporated by reference herein. Binding ofcell surface proteins, CD304, CD1c, CD40 and CD80 to the correspondingaptamers on sensors transduces an electrical signal that is relayed tothe controller of the microfluidic device. The controller in turnactuates pumps and valves to responsively interact with the APC. Forexample, binding of a myeloid dendritic cell to the CD1c sensor in bay 1signals the controller to actuate a pump and valve to inject GMCSF (atapproximately 10 μg/ml) and prostatic acid phosphatase antigen to cellbay 1 to promote differentiation, activation and antigen loading of theengaged dendritic cells. See Small et al., J. Clin. Oncol. 18:3894-3903, 2000 which is incorporated herein by reference. Activation ofthe dendritic cells in cell bay 1 is accompanied by increased CD80 andCD40 expression on the cell surface. Aptamer sensors in bay 1 bind toCD80 and CD40 and signal the controller that an activated dendritic cellis engaged in bay 1. The controller responds by enabling hydrofluidicflow to move the activated dendritic cell to cell bay 2. Activateddendritic cells are retained in bay 2 by binding to membrane boundanti-HLA-DR scFv and anti-CD54 scFv. Unactivated dendritic cells are notretained in bay 2 and routed to a waste reservoir. After a few minutesof fluid flow to move the dendritic cells from bay 1, a piezoelectricgate is opened to route the activated dendritic cells to a cellcollection trap, and cell bay 2 is flooded with media emanating from amicropump and valve to drive activated dendritic cells to a cell trap.Activated, antigen-loaded, APC are harvested from the cell trap andcharacterized. Activated, antigen-loaded APC are used for immunotherapyin patients and for immunology research.

Example 4 A Method to Treat a Patient with Prostate Cancer Using aMicropattern Cell Treatment Array Ex Vivo to Vaccinate the Patient withDendritic Cells and Prostate Cancer Antigens

A patient with hormone refractory prostate cancer is treated with immunecells derived from an ex vivo device including a micropattern array thatdifferentiates, activates, loads and collects antigen presenting cells(APC) for immune cell therapy. Dendritic cell (DC) precursors areobtained from the patient's peripheral blood and they are processed in adevice including the micropattern array that responsively interacts withthe cells to differentiate, activate, load and select them prior toreinfusion in the patient. Immunotherapy for prostate cancer withantigen-loaded dendritic cells is provided. See, e.g., Small et al., J.Clin. Oncol. 18: 3894-3903, 2000 and Gilboa, J. Clin. Invest. 117:1195-1203, 2007, which are incorporated herein by reference.

The patient has a prostatic acid phosphatase (PAP) level in his serumthat is greater than or equal to 2 times the upper limit of normal PAP(3.0 ng/mL) and has histologically confirmed adenocarcinoma of theprostrate. To obtain dendritic cell precursors, the patient's peripheralblood mononuclear cells (PBMNC) are collected by leukapheresis followedby density gradient centrifugation. See e.g., Small et al., Ibid.Dendritic cells are isolated from the PBMNC by methods as provided. See,e.g., Product Data Sheet: “Blood Dendritic Cell Isolation Kit II”available from Miltenyi Biotec Inc., Auburn, CA which is incorporatedherein by reference. PBMNC are subjected to depletion to remove B cellsand monocytes followed by positive selection with magnetic beads thatrecognize plasmacytoid DC and myeloid DC (i.e., conventional DC).Purified DC populations (approximately 90-98% pure) represent about 1%of total PBMNC. Purified DC precursor cells are injected into an entryport of a micropattern array device that distributes the cells tomultiple parallel microcircuits which responsively interact with thedendritic cell precursors to generate mature, antigen-loaded dendriticcells for reinfusion into the patient with prostate cancer.

The device including the micropattern array contains multiple(approximately 75) parallel micro-circuits that engage, differentiate,activate and load dendritic cells with antigen. Methods to isolatedendritic precursor cells from peripheral blood mononuclear cells(PBMNC) are provided. See, e.g., Angelot et al., Haematologica 94:1502-1512, 2009 which is incorporated herein by reference. Eachmicro-circuit includes an entry channel leading to cell bay 1, an exitchannel leading to entry to cell bay 2, and an exit channel leading to acell collection trap. Cell bay 1 contains: surface-bound CD1c sensors; agranulocyte macrophage colony stimulating factor (GMCSF) injector;surface bound CD304 sensors; a cytosine-guanosine (CpG)-richoligodeoxynucleotide (ODN) injector; an injector for a tumor-associatedantigen, prostate acid phosphatase (PAP); CD40 sensors; and CD80sensors. Cell bay 2 contains: membrane bound anti-CD304 scFv, membranebound anti-CD1c scFv, and membrane bound CD40 ligand. Cell bays areconstructed with a lipid bilayer containing embedded membrane proteinsand gold electrode regions bearing aptamer-based sensors. See e.g., Laiet al., Anal. Chem. 79: 229-233, 2007 which is incorporated herein byreference.

Dendritic precursor cells, that express CD1c (myeloid DC) or CD304(plasmacytoid DC) flow into cell bay 1 and bind to the CD1c sensor orCD304 sensor, respectively. Following myeloid DC binding to the CD1csensor, the sensor signals the controller to actuate a pump and valvethat provide GMCSF at approximately 10 μg/m1 and PAP antigen atapproximately 10 μg/mL to cell bay 1 to promote differentiation,activation and loading of engaged myeloid DC. Upon plasmacytoid DCbinding to the CD304 sensor a signal to the controller leads toactuation of a pump and valve that dispense CPG-ODN and PAP antigen.Activation and maturation of the dendritic cells in cell bay 1,indicated by CD80 and/or CD40 expression, is detected by thecorresponding aptamer sensors. Signaling from the CD40 or CD80 sensorsin bay 1 to the controller results in actuating a micro-pump to pumpfluid into bay 1 and opening an exit valve to allow the activated DC toflow into bay 2 of the device. Activated DC are retained in bay 2 bybinding to CD40 ligand while unactivated DC are not retained in bay 2and routed to a waste reservoir. After a few minutes of fluid flow tomove the DC from bay 1, a piezoelectric gate is opened to route theactivated DC to a cell collection trap. Cell bay 2 is flooded with mediaemanating from a micropump and valve to drive activated DC to the celltrap. The DC are harvested from the cell trap and characterized prior toreinfusion in the patient.

Activated DC processed in the device including the micropattern arrayare analyzed to verify that they are safe and effective APC fortreatment of prostate cancer in the patient. Methods to characterize APCfor infusion are provided. See, e.g., Small et al., Ibid.). A sterilitytest of the cell preparation is done by checking for microbial growthafter 40 hours in nutrient broth at 37° C. Endotoxin tests (criterion isless than 1.4 EU/ml) and mycoplasma testing are done. The activated DCare phenotyped by flow cytometry to assess maturation and activation. DCare stained with fluorescent antibodies (Ab) specific for: CD304, CD1c,HLA-DR, CD80, CD86 and CD40. CD304 and CD1c Ab are available fromMiltenyi Biotec Inc., Auburn, Calif.; HLA-DR, CD80, CD86 and CD40 Ab areavailable from BD Biosciences, San Jose, Calif.. Criteria foridentification of mature, activated DC are described. See e.g., Angelotet al., Ibid.

To evaluate the immune function of the processed DC, the activated DCare tested for their ability to stimulate allogeneic and autologous Tcells in a proliferation assay. Assays to test human DC in mixedlymphocyte reactions are provided. See, e.g., Small et al., Ibid. HumanT cells are isolated from the PBMNC of allogeneic donors or from thepatient (autologous) using CD3 T cell enrichment columns to provideallogeneic or autologous responder cells. Isolated DC precursor cells(pre-processing) or activated DC (post-processing) are compared incultures including tritiated thymidine to monitor T cell proliferationin culture. For both allogeneic and autologous T cells, activated DC(post-processing) stimulate 4-5 times the T cell proliferationstimulated by precursor DC (pre-processing).

Activated, mature DC loaded with PAP and tested for safety and immunefunction are formulated in lactated Ringers' solution and stored at 4°C. until they are infused in the patient with prostate cancer.Approximately 0.2×10⁸ to 2.0×10⁸ activated DC/m² are infused in totalvolume of 250 mL at 0, 4 and 8 weeks. Proliferation of the tumor ismonitored in the patient, and additional infusions of activated matureDC loaded with PAP can be prepared using the device including themicropattern array starting with frozen PBMNC from the patient orfreshly harvested PBMNC.

Each recited range includes all combinations and sub-combinations ofranges, as well as specific numerals contained therein.

All publications and patent applications cited in this specification areherein incorporated by reference to the extent not inconsistent with thedescription herein and for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference for all purposes.

The state of the art has progressed to the point where there is littledistinction left between hardware and software implementations ofaspects of systems; the use of hardware or software is generally (butnot always, in that in certain contexts the choice between hardware andsoftware can become significant) a design choice representing cost vs.efficiency tradeoffs. There are various vehicles by which processesand/or systems and/or other technologies described herein can beeffected (e.g., hardware, software, and/or firmware), and that thepreferred vehicle will vary with the context in which the processesand/or systems and/or other technologies are deployed. For example, ifan implementer determines that speed and accuracy are paramount, theimplementer may opt for a mainly hardware and/or firmware vehicle;alternatively, if flexibility is paramount, the implementer may opt fora mainly software implementation; or, yet again alternatively, theimplementer may opt for some combination of hardware, software, and/orfirmware. Hence, there are several possible vehicles by which theprocesses and/or devices and/or other technologies described herein maybe effected, none of which is inherently superior to the other in thatany vehicle to be utilized is a choice dependent upon the context inwhich the vehicle will be deployed and the specific concerns (e.g.,speed, flexibility, or predictability) of the implementer, any of whichmay vary. Optical aspects of implementations will typically employoptically-oriented hardware, software, and/or firmware.

In a general sense the various aspects described herein which can beimplemented, individually and/or collectively, by a wide range ofhardware, software, firmware, or any combination thereof can be viewedas being composed of various types of “electrical circuitry.”“Electrical circuitry” includes, but is not limited to, electricalcircuitry having at least one discrete electrical circuit, electricalcircuitry having at least one integrated circuit, electrical circuitryhaving at least one application specific integrated circuit, electricalcircuitry forming a general purpose computing device configured by acomputer program (e.g., a general purpose computer configured by acomputer program which at least partially carries out processes and/ordevices described herein, or a microprocessor configured by a computerprogram which at least partially carries out processes and/or devicesdescribed herein), electrical circuitry forming a memory device (e.g.,forms of random access memory), and/or electrical circuitry forming acommunications device (e.g., a modem, communications switch, oroptical-electrical equipment). The subject matter described herein maybe implemented in an analog or digital fashion or some combinationthereof.

The herein described components (e.g., steps), devices, and objects andthe description accompanying them are used as examples for the sake ofconceptual clarity and that various configuration modifications usingthe disclosure provided herein are within the skill of those in the art.Consequently, the specific examples set forth and the accompanyingdescription are intended to be representative of their more generalclasses. In general, use of any specific example herein is also intendedto be representative of its class, and the non-inclusion of suchspecific components (e.g., steps), devices, and objects herein shouldnot be taken as indicating that limitation is desired.

With respect to the use of substantially any plural or singular termsherein, the reader can translate from the plural to the singular or fromthe singular to the plural as is appropriate to the context orapplication. The various singular/plural permutations are not expresslyset forth herein for sake of clarity.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely examples, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected,” or“operably coupled,” to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable,” to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable or physically interacting componentsor wirelessly interactable or wirelessly interacting components orlogically interacting or logically interactable components.

While particular aspects of the present subject matter described hereinhave been shown and described, changes and modifications may be madewithout departing from the subject matter described herein and itsbroader aspects and, therefore, the appended claims are to encompasswithin their scope all such changes and modifications as are within thetrue spirit and scope of the subject matter described herein.Furthermore, it is to be understood that the invention is defined by theappended claims. It will be understood that, in general, terms usedherein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood that if a specific number of anintroduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to inventions containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an”; the same holdstrue for the use of definite articles used to introduce claimrecitations. In addition, even if a specific number of an introducedclaim recitation is explicitly recited, such recitation should typicallybe interpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, typicallymeans at least two recitations, or two or more recitations).Furthermore, in those instances where a convention analogous to “atleast one of A, B, and C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, and C”would include but not be limited to systems that have A alone, B alone,C alone, A and B together, A and C together,

B and C together, or A, B, and C together, etc.). In those instanceswhere a convention analogous to “at least one of A, B, or C, etc.” isused, in general such a construction is intended in the sense one havingskill in the art would understand the convention (e.g., “a system havingat least one of A, B, or C” would include but not be limited to systemsthat have A alone, B alone, C alone, A and B together, A and C together,B and C together, or A, B, and C together, etc.). Virtually anydisjunctive word and/or phrase presenting two or more alternative terms,whether in the description, claims, or drawings, should be understood tocontemplate the possibilities of including one of the terms, either ofthe terms, or both terms. For example, the phrase “A or B” will beunderstood to include the possibilities of “A” or “B” or “A and B.”

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is: 1-141. (canceled)
 142. A device, comprising: aquasi-planar substrate; one or more cell bays on the quasi-planarsubstrate, each cell bay including one or more fixed effector moleculesand configured to interact with one or more cells; at least one channelin fluid communication with the one or more cell bays and configured forproviding passage of one or more cells proximate the one or more cellbays; at least one gate or valve coupled to at least one of the one ormore cell bays and configured to provide passage of the one or morecells to at least one cell trap in the one or more cell bays; one ormore sensors configured to detect at least one of binding or releasefrom binding between the one or more fixed effector molecules and theone or more cells; at least one controller in communication with the oneor more sensors configured to control at least one of the gate or valve,or the fluid channel, in response to the sensed binding or release ofone or more cells from the one or more fixed effector molecules. 143.The device of claim 142, wherein the one or more cells include at leastone of a dendritic cell, stem cell, lymphocyte or NK cell.
 144. Thedevice of claim 143, wherein the stem cell includes at least one of ahematopoietic stem cell, myeloid stem cell, or pluripotent stem cell.145. The device of claim 142, wherein the one or more cells include atleast one of a Treg cell.
 146. The device of claim 142, wherein the oneor more fixed effector molecules include at least one of an antigen,major histocompatibility complex (MHC), antigen-loaded WIC,co-stimulatory molecule, or cytokine.
 147. The device of claim 146,wherein the antigen includes at least one of a tumor-associated antigen,or viral peptide antigen.
 148. The device of claim 142, wherein the oneor more fixed effector molecules include at least one of an integralmembrane protein, glycophosphatidylinositol-anchored protein, fusionprotein, conjugated protein, aptamer or polymer.
 149. The device ofclaim 142, wherein the one or more sensors include at least one of athermal detector, an electrical detector, a chemical detector, anoptical detector, an ion detector, a biological detector, a radioisotopedetector, an electrochemical detector, a radiation detector, an acousticdetector, a magnetic detector, a capacitive detector, a pressuredetector, an ultrasonic detector, an infrared detector, a microwavemotion detector, a radar detector, an electric eye, an image sensor.150. The device of claim 142, further including at least one micropumpoperably coupled to the controller.
 151. The device of claim 150,wherein the at least one micropump is configured to increase the mediumlevel in a cell bay to assist in movement of the cell out of the cellbay.
 152. The device of claim 142, wherein the membrane structureincludes at least one of a lipid bilayer or a lipid monolayer.